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1.
Spontaneous peptide bond cleavage in aging alpha-crystallin through a succinimide intermediate 总被引:4,自引:0,他引:4
C E Voorter W A de Haard-Hoekman P J van den Oetelaar H Bloemendal W W de Jong 《The Journal of biological chemistry》1988,263(35):19020-19023
Cleavage of specific peptide bonds occurs with aging in the alpha A subunit of bovine alpha-crystallin. One of the breaks occurs at residue Asn-101. This same residue undergoes in vivo deamidation, isomerization, and racemization. Deamidation and isomerization are known to occur via succinimide ring formation of labile asparagine residues. Model studies on peptides have shown that imide formation can also lead to peptide bond cleavage (Geiger, T., and Clarke, S. (1987) J. Biol. Chem. 262, 785-794). In that case, both asparagine and aspartic acid amide would be expected as C termini of the truncated polypeptide, and this is indeed the case in the alpha A-(1-101)-chain. This thus represents a first example of nonenzymatic in vivo peptide bond cleavage in an aging protein through the formation of a succinimide intermediate. In addition, we found that in bovine lens no detectable conversion (through the action of protein-carboxyl methyltransferase) of isoaspartyl to normal aspartyl residues occurs in vivo after deamidation of Asn-101. 相似文献
2.
Evidence for the in vivo deamidation and isomerization of an asparaginyl residue in cytosolic serine hydroxymethyltransferase 总被引:6,自引:0,他引:6
Rabbit liver cytosolic serine hydroxymethyltransferase exists in several subforms which have different isoelectric points. Incubation of the purified enzyme with chymotrypsin cleaves the enzyme at Trp14. The released amino-terminal 14-mer peptide was shown to exist in three forms of equal concentration. The peptides differ in structure only at the asparaginyl residue at position 5. In addition to asparagine at this position we found both aspartyl and isoaspartyl residues. The deamidation of Asn5 does not appear to occur during the purification of the enzyme. The in vitro rate of deamidation of Asn5 in the enzyme is more than 5-fold slower than the rate of deamidation of this residue in the free 14-mer peptide. The isoaspartyl residue at position 5 serves as a substrate for protein carboxyl methyltransferase both in the free 14-mer peptide and the native enzyme. The enzyme which has had the amino-terminal 14 residues removed by digestion with chymotrypsin still exists in several forms with different isoelectric points. Reaction of peptides from this enzyme with carboxyl methyltransferase suggests that there is at least one more asparaginyl residue in this enzyme other than Asn5 which has undergone deamidation with the formation of isoaspartyl bonds. 相似文献
3.
Enzymatic protein carboxyl methylation at physiological pH: cyclic imide formation explains rapid methyl turnover 总被引:5,自引:0,他引:5
At pH 7.4, 37 degrees C, bovine brain protein carboxyl methyltransferase transiently methylates deamidated adrenocorticotropin. The methylation occurs at the alpha-carboxyl group of an atypical beta-carboxyl-linked isoaspartyl residue (position 25). Several lines of evidence indicate that the immediate product of demethylation is an aspartyl cyclic imide involving positions 25 and 26. The evidence includes (1) the rapid rate of methyl ester hydrolysis, which is consistent with intramolecular catalysis, (2) the inability of the demethylated product to be remethylated, (3) the charge of this product, and (4) its rate of breakdown. The eventual hydrolysis of the cyclic imide produces a 30/70 mixture of peptides containing either alpha- or beta-carboxyl-linked aspartyl residues, respectively. Cyclic imide formation is nonenzymatic and can explain the unusual lability of mammalian protein methyl esters in general. These findings suggest that protein carboxyl methylation in mammalian tissues is not a simple on/off reversible modification as it apparently is in chemotactic bacteria. Carboxyl methylation may serve to activate selected protein carboxyl groups for subsequent longer lasting modifications, possibly subserving a role in protein repair, degradation, cross-linking, or some other as yet undiscovered alteration of protein structure. 相似文献
4.
5.
Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A. 总被引:3,自引:1,他引:2
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F. Catanzano G. Graziano S. Capasso G. Barone 《Protein science : a publication of the Protein Society》1997,6(8):1682-1693
Selective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature-induced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. Differential scanning calorimetry measurements were performed in the pH range 3.0-6.0, where the unfolding process is reversible, according to the reheating criterion used. It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In particular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 degrees C of denaturation temperature and 65 kJ mol-1 of denaturation enthalpy at pH 5.0. These results are discussed and correlated to the X-ray three-dimensional structure of this derivative. The analysis leads to the conclusion that the difference in thermal stability between RNase A and (N67isoD)RNase A is due to enthalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entropic effects. Finally, the influence of cytidine-2'-monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins. 相似文献
6.
Repair of isopeptide bonds by protein carboxyl O-methyltransferase: seminal ribonuclease as a model system 总被引:8,自引:0,他引:8
Previous work has shown that in the peptide segment 62-76 of naturally deamidated alpha subunit of bovine seminal ribonuclease (BS-RNase) the alpha-carboxyl group of iso-Asp67 is selectively methylated by S-adenosylmethionine:protein carboxyl O-methyltransferase [Di Donato, A., Galletti, P., & D'Alessio, G. (1986) Biochemistry 25, 8361-8368]. In the present study this reaction has been characterized, by using the tryptic segment 62-76 of the protein chain (peptide alpha 16). The peptide is stoichiometrically methyl esterified with a Km of 6.17 microM and a Vmax of 19.56 nmol min-1 mg-1, and the product of demethylation has been identified as the cyclic succinimidyl derivative of iso-Asp67-Gly68. The cleavage of the succinimidyl ring yields two isomeric peptides containing an aspartyl residue (peptide alpha 17) and an isoaspartyl residue (peptide alpha 16). On the basis of these results conditions were defined in which repeated cycles of methylation-demethylation led to an effective conversion of peptide alpha 16 into peptide alpha 17, a process that can be interpreted as the repair of an altered isopeptide bond. When the methyl esterification reaction was studied on the native dimeric isoenzymes of seminal RNase and on catalytically active monomeric derivatives, including a stabilized alpha-type subunit, the results of these experiments showed that none of the protein forms were substrates for the methyltransferase. Only the unfolded alpha-type subunit was methylated to a stoichiometric extent. These results indicate that the repair of altered isopeptide bonds is chemically feasible in peptides but is hindered in the case of seminal RNase by its three-dimensional structure. 相似文献
7.
M Xie J Aubé R T Borchardt M Morton E M Topp D Vander Velde R L Schowen 《The journal of peptide research》2000,56(3):165-171
Mimetics of beta-turn structures in proteins have been used to calibrate the relative reactivities toward deamidation of asparagine residues in the two central positions of a beta-turn and in a random coil. N-Acetyl-Asn-Gly-6-aminocaproic acid, an acyclic analog of a beta-turn mimic undergoes deamidation of the asparaginyl residue through a succinimide intermediate to generate N-acetyl-Asp-N-Gly-6-aminocaproic acid (6-aminocaproic acid, hereafter Aca) and N-acetyl-L-iso-aspartyl (isoAsp)-Gly-Aca (pH 8.8, 37 degrees C) approximately 3-fold faster than does the cyclic beta-turn mimic cyclo-[L-Asn-Gly-Aca] with asparagine at position 2 of the beta-turn. The latter compound, in turn, undergoes deamidation approximately 30-fold faster than its positional isomer cyclo-[Gly-Asn-Aca] with asparagine at position 3 of the beta-turn. Both cyclic peptides assume predominantly beta-turn structures in solution, as demonstrated by NMR and circular dichroism characterization. The open-chain compound and its isomer N-acetyl-Gly-Asn-Aca assume predominantly random coil structures. The latter isomer undergoes deamidation 2-fold slower than the former. Thus the order of reactivity toward deamidation is: asparagine in a random coil approximately 3x(asparagine) in position 2 of a beta-turn approximately 30x (asparagine) in position 3 of a beta-turn. 相似文献
8.
Athmer L Kindrachuk J Georges F Napper S 《The Journal of biological chemistry》2002,277(34):30502-30507
Proteins are vulnerable to spontaneous, covalent modifications that may result in alterations to structure and function. Asparagines are particularly labile, able to undergo deamidation through the formation of a succinimide intermediate to produce either aspartate or isoaspartate residues. Although aspartates cannot undergo deamidation they can form a succinimide and result in the same products. Isoaspartyls are the principal product of succinimide hydrolysis, accounting for 65-85% of the emerging residues. The variability in the ratio of products emerging from succinimide hydrolysis suggests the ability of protein structure to influence succinimide outcome. In the H15D histidine-containing protein (HPr), phosphorylation of the active site aspartate catalyzes the formation of a cyclic intermediate. Resolution of this species is exclusively to aspartate residues, suggestive of either a succinimide with restrained hydrolysis, or an isoimide, from which aspartyl residues are the only possible product. Deletion of the C-terminal residue of this protein does not influence the ability for phosphorylation or ring formation, but it does allow for isoaspartyl formation, verifying a succinimide as the cyclic intermediate in H15D HPr. Isoaspartyl formation in H15D Delta85 is rationalized to occur as a consequence of elimination of steric restrictions imposed by the C terminus on the main-chain carbonyl of the succinimide, the required point of nucleophilic attack of a water molecule for isoaspartyl formation. This is the first reported demonstration of the influence of protein structure on the products emerging from succinimide hydrolysis. 相似文献
9.
J E Walker I M Fearnley N J Gay B W Gibson F D Northrop S J Powell M J Runswick M Saraste V L Tybulewicz 《Journal of molecular biology》1985,184(4):677-701
The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
10.
Formation of isoaspartate at two distinct sites during in vitro aging of human growth hormone 总被引:4,自引:0,他引:4
B A Johnson J M Shirokawa W S Hancock M W Spellman L J Basa D W Aswad 《The Journal of biological chemistry》1989,264(24):14262-14271
In vitro aging at pH 7.4, 37 degrees C causes natural sequence recombinant human growth hormone (rhGH), methionyl rhGH, and human pituitary growth hormone to become substrates for bovine brain protein carboxyl methyltransferase, an enzyme that modifies the "side chain" alpha-carboxyl group present at atypical isoaspartyl linkages. The substrate capacity of rhGH increased at a rate of 1.8 methyl-accepting sites/day/100 molecules of hormone. Reversed-phase high performance liquid chromatography (HPLC) of trypsin digests of aged rhGH revealed two altered peptides not present in digests of control rhGH. These two fragments, which had the amino acid compositions of residues 128-134 (Leu-Glu-Asp-Gly-Ser-Pro-Arg) and 146-158 (Phe-Asp-Thr-Asn-Ser-His-Asn-Asp-Asp-Ala-Leu-Leu-Lys), contained the majority of the induced methylation sites, 22 and 58%, respectively. Isoaspartate can result from deamidation of asparagine or isomerization of aspartate. Isomerization of Asp-130, the only candidate site in 128-134, was corroborated by coelution of the altered fragment with the synthetic isoaspartyl peptide upon reversed-phase HPLC. Evidence is presented that the altered 146-158 fragment is a mixture of two peptides resulting from deamidation of Asn-149 to form 70-80% isoaspartate and 20-30% aspartate at this position. The position of isoaspartate in the altered 146-158 fragment was deduced from mass spectrometry, which indicated a single deamidated asparagine; from methylation stoichiometry, which indicated only one methylation site; and from automated Edman degradation, which showed an absence of asparagine and a low yield of aspartate at position 149. These results show that isoaspartate formation from both aspartate and asparagine is a significant, and possibly the major, source of spontaneous covalent alteration of rhGH and that enzymatic carboxyl methylation provides a powerful tool for assessing this type of modification. 相似文献
11.
Esposito L Vitagliano L Sica F Sorrentino G Zagari A Mazzarella L 《Journal of molecular biology》2000,297(3):713-732
Crystals of the deamidated form of bovine pancreatic ribonuclease which contains an isoaspartyl residue in position 67 diffract to 0. 87 A at 100 K. We have refined the crystallographic model using anisotropic displacement parameters for all atoms to a conventional crystallographic residual R=0.101 for all observed reflections in the resolution range 61.0-0.87 A. The ratio observations/parameters is 7.2 for the final model. This structure represents one of the highest resolution protein structures to date and interestingly, it is the only example containing more than one molecule in the asymmetric unit with a resolution better than 1.0 A. The non-crystallographic symmetry has been used as a validation check of the geometrical parameters and it has allowed an estimate for an upper limit of errors associated with this high resolution model. In the present structure it was possible to obtain a more accurate picture of the active site whose electron density was not clearly interpretable in the previous 1.9 A resolution structure. In particular, the P1 site is alternatively occupied either by a sulphate anion or by a water molecule network. Most of hydrogen atoms were visible in the electron density maps, including those involved in C(alpha)-H(alpha).O interactions. Analysis of protein-solvent interactions has revealed the occurrence of an extensive cluster of water molecules, predominantly arranged in pentagonal fused rings and surrounding hydrophobic moiety of side-chains. Finally, in spite of the limited sample of residues, we have detected a clear dependence of backbone N-C(alpha)-C angle on residue conformation. This correlation can be fruitfully used as a valuable tool in protein structure validation. 相似文献
12.
Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation 总被引:27,自引:0,他引:27
Aspartyl and asparaginyl deamidation, isomerization, and racemization reactions have been studied in synthetic peptides to model these spontaneous processes that alter protein structure and function. We show here that the peptide L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala undergoes a rapid deamidation reaction with a half-life of only 1.4 days at 37 degrees C, pH 7.4, to give an aspartyl succinimide product. Under these conditions, the succinimide product can further react by hydrolysis (half-time, 2.3h) and by racemization (half-time, 19.5 h). The net product of the deamidation reaction is a mixture of L- and D-normal aspartyl and beta-transpeptidation (isoaspartyl) hexapeptides. Replacement of the asparagine residue by an aspartic acid residue results in a 34-fold decrease in the rate of succinimide formation. Significant racemization was found to accompany the deamidation and isomerization reactions, and most of this could be accounted for by the rapid racemization of the succinimide intermediate. Replacement of the glycyl residue in the asparagine-containing peptide with a bulky leucyl or prolyl residue results in a 33-50-fold decrease in the rate of degradation. Peptide cleavage products are observed when these Asn-Leu and Asn-Pro-containing peptides are incubated. Our studies indicate that both aspartic acid and asparagine residues may be hot spots for the nonenzymatic degradation of proteins, especially in cells such as erythrocytes and eye lens, where these macromolecules must function for periods of about 120 days and 80 years, respectively. 相似文献
13.
14.
Structural basis for ligand selectivity of heteromeric olfactory cyclic nucleotide-gated channels
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In vertebrate olfactory receptors, cAMP produced by odorants opens cyclic nucleotide-gated (CNG) channels, which allow Ca(2+) entry and depolarization of the cell. These CNG channels are composed of alpha subunits and at least two types of beta subunits that are required for increased cAMP selectivity. We studied the molecular basis for the altered cAMP selectivity produced by one of the beta subunits (CNG5, CNCalpha4, OCNC2) using cloned rat olfactory CNG channels expressed in Xenopus oocytes. Compared with alpha subunit homomultimers (alpha channels), channels composed of alpha and beta subunits (alpha+beta channels) were half-activated (K(1/2)) by eightfold less cAMP and fivefold less cIMP, but similar concentrations of cGMP. The K(1/2) values for heteromultimers of the alpha subunit and a chimeric beta subunit with the alpha subunit cyclic nucleotide-binding region (CNBR) (alpha+beta-CNBRalpha channels) were restored to near the values for alpha channels. Furthermore, a single residue in the CNBR could account for the altered ligand selectivity. Mutation of the methionine residue at position 475 in the beta subunit to a glutamic acid as in the alpha subunit (beta-M475E) reverted the K(1/2,cAMP)/K(1/2,cGMP) and K(1/2, cIMP)/K(1/2,cGMP) ratios of alpha+beta-M475E channels to be very similar to those of alpha channels. In addition, comparison of alpha+beta-CNBRalpha channels with alpha+beta-M475E channels suggests that the CNBR of the beta subunit contains amino acid differences at positions other than 475 that produce an increase in the apparent affinity for each ligand. Like the wild-type beta subunit, the chimeric beta/alpha subunits conferred a shallow slope to the dose-response curves, increased voltage dependence, and caused desensitization. In addition, as for alpha+beta channels, block of alpha+betaCNBRalpha channels by internal Mg(2+) was not steeply voltage-dependent (zdelta approximately 1e(-)) as compared to block of alpha channels (zdelta 2.7e(-)). Thus, the ligand-independent effects localize outside of the CNBR. We propose a molecular model to explain how the beta subunit alters ligand selectivity of the heteromeric channels. 相似文献
15.
Deamidation of the asparaginyl-glycyl sequence 总被引:2,自引:0,他引:2
Y C Meinwald E R Stimson H A Scheraga 《International journal of peptide and protein research》1986,28(1):79-84
The deamidation of Ac-Asn-Gly-NHMe and Ac-Isn-Gly-NHMe has been studied as a model for the facile deamidation of the Asn-Gly sequence in proteins. At alkaline pH, the product in each case is an identical mixture of Ac-alpha-Asp-Gly-NHMe (approximately 22%) and Ac-beta-Asp-Gly-NHMe (approximately 78%) as determined by n.m.r. spectroscopy. Because this same ratio is obtained from both Ac-Asn-Gly-NHMe and Ac-Isn-Gly-NHMe, the postulated mechanism, that deamidation proceeds through a cyclic imide intermediate, is confirmed. Unlike peptides of aspartyl esters, cyclization does not occur under nonaqueous conditions or at low pH in aqueous solution. 相似文献
16.
Garino C Bihel F Souard F Quéléver G Kraus JL 《Bioorganic & medicinal chemistry letters》2004,14(7):1771-1774
During the coupling reaction between 3-alkoxy-7-amino-4-chloroisocoumarin and N-acyl alanine dipeptide, an unexpected deamidation reaction was observed. The proposed mechanism for this reaction involved the formation of an imide intermediate which after cleavage led to the release of amino acid moiety. The described deamidation reaction represents the first chemical model involving a non-peptidic moiety, which mimics biological and chemical deamidation processes occurring in proteins or peptides incorporating an asparagine or a glutamine residue. 相似文献
17.
P T Wingfield R J Mattaliano H R MacDonald S Craig G M Clore A M Gronenborn U Schmeissner 《Protein engineering》1987,1(5):413-417
Recombinant-derived human interleukin-1 alpha (IL-1 alpha), purified from Escherichia coli, was resolved by isoelectric focusing on polyacrylamide gels into two species of isoelectric points (pI) 5.45 and 5.20, which constituted approximately 75% and approximately 25% of the total IL-1 alpha protein respectively. The pI 5.45 and pI 5.20 species were separated by chromatofocusing and subjected to N-terminal sequence analysis. The pI 5.45 species contained the expected Asn residue at position 36 of the mature protein sequence whereas the pI 5.20 species contained an Asp residue at the same position. A mutant protein in which Asn-36 was substituted for a Ser residue was isolated from E. coli and shown to be homogeneous on isoelectric focusing analysis with a pI = 5.45. 1H-n.m.r. and circular dichroism analyses of wild-type and the mutant IL-1 alpha indicated a similar conformation which was also indicated by the identical receptor binding affinities of IL-1 alpha with Asn, Asp or Ser in position 36. The mutant protein was stabilized against specific base-catalysed and temperature-induced deamidation, and may be more suitable than the wild-type position for physical and structural studies. 相似文献
18.
1) A reproducible procedure was developed for the purification of bovine follitropin. 2) The method involved ammonium sulfate precipitation, ion exchange and adsorption chromatography, concanacaline-A-Sepharose chromatography and gel filtration. 3) A specific radioligand receptor assay was used to monitor each chromatographical step. 4) The potency of highly purified bovine follitropin as measured by Steelman and Pohley bioassay was 62 times the NIH-FSH-B1 standard preparation. 5) Contaminations of bovine follitropin by other glycoprotein hormones such as thyrotropin and lutropin amounted to 3 and 0.45 per cent by weight respectively as measured by specific radioimmunoassays and radioligand receptor assays. 6) The subunits alpha and beta of bovine follitropin were obtained by incubation in acidic urea, the chains being then separated by anion exchange chromatography. The subunits were subjitted to complete characterization. The amino-terminal residue of the alpha subunit is phenylalanine while a half cystine residue was found at the aminoterminal end of the beta chain. 8) Cross-contamination of the alpha and beta subunit preparations was measured by specific radioimmunoassays and amounted to 0.02 and 0.1 per cent by weight respectively. 相似文献
19.
We have found that a chicken egg lysozyme derivative (beta-101-lysozyme) containing an L-isoaspartyl residue at position 101 has a Km for methylation by the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77) of 183 microM, about 30 times higher than that expected from previous studies with isoaspartyl-containing peptides. In the course of investigating the reasons for this poor enzyme recognition, we found that charged residues on the carboxyl side of isoaspartyl residues had a large effect on the affinity of the enzyme for synthetic peptides. This is best illustrated by the lysozyme-related peptide YVSisoDGDG, which has a Km for methylation of 469 microM. When the penultimate aspartyl residue is replaced by a cysteinyl residue, the Km drops to 4.6 microM, comparable to other peptides of similar size. Furthermore, replacing it with a cysteic acid residue results in a Km of 104 microM, suggesting that a negative charge at this position may lead to a weaker affinity of the peptide substrate for the methyltransferase. Assays with additional synthetic peptides indicate that moving the negative charge to the first or third residue on the carboxyl side of the isoaspartyl residue has a similar but less severe effect in reducing its affinity for the methyltransferase. Enzymatic methylation has recently been proposed to be the first step in the conversion of abnormal isoaspartyl residues to aspartyl residues. The results reported here, however, along with previous evidence that protein tertiary structure can inhibit isoaspartyl methylation, suggest that only a subclass of damaged sites are capable of efficiently entering a putative repair pathway; the sites not recognized by the methyltransferase may accumulate in vivo. 相似文献
20.
T A Neubert R S Johnson J B Hurley K A Walsh 《The Journal of biological chemistry》1992,267(26):18274-18277
Rod transducin (Tr), a heterotrimeric GTP-binding protein composed of alpha, beta, and gamma subunits, couples photolysis of rhodopsin to the activation of cyclic GMP phosphodiesterase in the vertebrate visual signal transduction cascade. To determine if T alpha r is covalently modified, we analyzed tryptic fragments of bovine retinal T alpha r using electrospray mass spectrometry, liquid chromatography/mass spectrometry, tandem mass spectrometry, and gas chromatography. A novel heterogeneous fatty acylation was detected at the NH2 terminus. Four types of NH2-terminal tryptic fragments of T alpha r were isolated, and each contained either a lauroyl (C12:0), myristoyl (C14:0), (cis-delta 5)-tetradecaenoyl (C14:1) or (cis,cis-delta 5, delta 8)-tetradecadienoyl (C14:2) fatty acyl residue amide-linked to the NH2-terminal glycine residue. NH2-terminal fatty acylation does not anchor T alpha r permanently in the membrane, since T alpha r used in these experiments was eluted without detergent from rod outer segment membranes. 相似文献